Method of sterilizing with alkylene oxide



' penser.

United States Patent Ofi" 3,169,906 Patented Feb. 16, 1965 ICC 3,169,906 METHOD OF STERILIZING WITH ALKYLENE OXIDE sterilants and food preservatives. Alkylene oxides, however, have not been used successfully as a sterilant for topical pharmaceutical preparations because of their highly toxic nature. For example, exposure of human skin for about two hours to a 1% solution of ethylene oxide in water will cause second degree burns (Arch. Indus. Hyg. Occup. Med. 2: 549-564, November 1950).

It has now been found that minute non-toxic concentrations in the range of 20 to 500 p.p.m. and preferably 20 to 200 p.p.m. of alkylene oxide in combination with an inert gas will successfully destroy even the most resistant microorganisms if exposed over a period of 2 to 60 days. It has further been found that if the mixture of alkylene oxide and inert gas is combined with the material to be sterilized and held under a pressure of 15 to 100 p.s.i.g. that the time in which sterility is achieved is substantially reduced. These findings not only provide a more economical means of sterilizing topical pharmaceutical preparations packaged in the form of an aerosol spray. but also provide a means for sterilizing those preparations or formulations which are not stable at the high temperatures used in autoclaving. Another advantage of the present invention is the fact that adequate proportions of alkylene oxide are retained within the pressurized dispenser throughout its use to prevent any contamination of the contents.

The alkylene oxides useful in this invention are ethylene oxide and propylene oxide, particularly useful, however, is ethylene oxide which compound is considered the more active sterilant.

The alkylene oxides are preferably blended with an inert gaseous propellant such as carbon dioxide or nitrogen. Particularly useful, arethe commercially available Freon and Genitron propellants such as monochlorodifluroethane, trichloromonofluromethane and mixtures thereof. The alkylene oxide is mixed with the propellant by introducing 40 to 1000 p.p.m. of alkylene oxide into a charged or liquified' cylinder of said propellant. Then the propellant-alkylene oxide mixture, normally in a liquid state, is introduced into the individual aerosol dispenser containing the topical pharmaceutical preparation and sealed therein under an internal pressure ,pf 15 to 100 p.s.i.g. by conventional means. The alkylene oxide-propellant mixture will effectively sterilize the aerosol preparation in 2 to 60 days, the actual time for any particular preparation will be dependent upon the concentration of alkylene oxide and internal pressure of the aerosol dis- 4 to 5 weeks under normal aerosol Even at minimum concentrations of about 20 p.p.m. complete sterilization can be expected within about 2 The following example is illustrative of the present in vention:

Example I A. To determine the effectiveness of minute non-toxic concentrations of ethylene oxide as a sterilant, a topical pharmaceutical preparation having the following formulation was prepared:

Percent Hexylene glycol 24.87 5 Polyethylene glycol 24:895 Furacin (5-nitro-2-furaldehyde semicarbazone) .21 Trichloromonofluromethane 25.00

Monochlorodifiuroethane-ethylene oxide mixture- 25.00

The aerosol units to be tested were inoculated with spores of Bacillus subtilis. Inoculum 5,625,000 cells/ml. of the above formulation. (Spore count was determined by plate dilution method using heart infusion agar and distilled Water pH 5.7.) Bacillus subtilis spores were selected because of their known resistance to the antimicrobial agentFuracin. After inoculation, the test units were filled with 1.25 oz. avoir. of the above glycol-Furacin solution then the 25% by weight of trichloromonofluromethane was added followed by addition of 25% by weight of the monochlorodifluromethane ethylene oxide mixture. The monochlorodifluroethane-ethylene oxide mixtures were prepared via a pressure burette such that the total concentration in the test units was varied from 500 to 0 p.p.m. The 0 p.p.m. being used as a control. Initial pressure within the test units was 18 p.s.i.g.

B. The above samples were tested for sterility at regular intervals by both the U81. and the Millipore Filter Technique with the following results:

(.1) U .S.P. sterility test pr0cea'ure. One second bursts (0.5 ml.) of Furacin Solution Aerosol from the inoculated sample was sprayed directly into 200 ml. thioglycollate broth. Flasks were then incubated at 37 C. for 72 hours. Aerosol containers were stored at 25 C. throughout the experiment. All samples indicating growth were checked by microscopic examination.

Formulation Non-sterile 1 Non-sterile l 4 4 4 0 Non-sterile l Formulation A-as given above. Formulation Bas given above but without .2% Furacin. 1 Non-sterilegrowth observed after 9 weeks incubation at 25 C.

, od, one 'cc. of the inoculated sample was sprayed into a Millipore filter and washed with one liter of sterile distilledwater. In this way, the Furacin was washed through; the filter leaving B. subtilz's spores on the filter. The filter was transferred to thioglycollate media'and incubated for 72 hours at 37 C. and checked for sterility. The units containing 500; 100 and 50 ppm. ethylene oxide were sterile; the unit containing p.p.m. ethylene oxide was.

non-sterile.

Example 11 To further illustrate. thepresent invention 115 0 aerosol samples containing Formulation A were prepared under non-sterile commercial packaging conditions by first preparing and filling the sample units with 1.25 oz. avoir.-of the 0.2% Furacin in hexylene glycol-polyethylene glycol base and then introducing the propellant (2, 5 by weight of trichloromonofiuromethane was added followed by addition of 25% by Weight of a monochlorodifluromethane- J ethylene oxide mixture) containing 70 ppm. ethylene oxide, such that the final concentration of ethylene oxide within the aerosol was.35 p.p.'m.

A total of 30 samples were removed from this run (10 from the beginning 10 from the-middle and 10 from the end) and checked fo r sterility by the Millipore Filter Technique. The samples were incubated at 37 C. and

55 C. for. 10 days using Difco Fluid Thioglycollate media. All samples were found to be sterile after 10 days.

What is claimedis:

1. In the methodof sterilizing with alkylene oxides is a substantially non-aqueous medium, the improvement by which human topical aerosol preparations are sterilized by protracted storage at room temperature which comprises charging said aerosol preparations under an internal pressure of to 110 p.s.i.g. with a gaseous mixture of an inert gaseous propellant selected from the group con- 'sisting of carbon dioxide, nitrogen andhalogenated lower alkyl' hydrocarbons containing at least one fluorine atom and an alkylene oxide containing 2 to 3 carbon atoms,

such that the sterilizing concentration of the alkylene oxide in the aerosol preparation will be in the rangeof to 500 p.p.m.; and storing said aerosol preparation under said pressure at room temperature for a period of 1 to 4 weeks. a 2. The method according to claim 1 wherein said alkyl ene oxide is ethylene oxide. 7

3. The method according to claim 1 wherein said alkylene oxide constitutes 40-1 000 p.p.m. of the gaseous mix- I ture. r

4. The method according to claim 1, wherein said alkylene oxide in'the aerosol preparation ispresent in the range of 20 to 200 p.p;m. and said aerosol is stored at room temperature for a period of 2 to 4 weeks.

References Cited by the Examiner UNITED STATES PATENTS 2,075,845 4/37 Grossjet'al. 21-58 i 2,705,696 4/55 Wolf et al; 16778 4/57 Kaye 16739 V 7 OTHER REFERENCES Chemical Abstracts i, vol. '44, pp. 6577i-d (1950), au-

' stract of Phillips et al., Am. J. Hyg. 50, 270-9, (1949).

- Chemical Abstracts, vol. 50, p. 17,256b (1956),. ab-' stract of Weichardt et a1., Berufsdermatosen, 4, 174-8, 

1. IN THE METHOD OF STERILIZING WITH ALKYLENE OXIDES IS A SUBSTANTIALLY NON-AQUEOUS MEDIUM, THE IMPROVEMENT BY WHICH HUMAN TOPICAL AEROSOL PREPARATIONS ARE STERILIZED BY PROTACTED STORAGE AT ROOM TEMPERATURE WHICH COMPRISES CHARGING SAID AEROSOL PREPARATIONS UNDER AN INTERNAL PRESSURE OF 15 TO 110 P.S.I.G. WITH A GASEOUS MIXTURE OF AN INERT GASEOUS PROPELLANT SELECTED FROM THE GROUP CONSISTING OF CARBON DIOXIDE, NITROGEN AND HALOGENATED LOWER ALKYL HYDROCARBONS CONTAINING AT LEAST ONE FLUORINE ATOM AND AN ALKYLENE OXIDE CONTAINING 2 TO 3 CARBON ATOMS, SUCH THAT THE STERILIZING CONCENTRATION OF THE ALKYLENE OXIDE INTHE AEROSOL PREPARATIONWILL BE IN THE RANGE OF 20 TO 500 P.P.M.; AND STORING SAID AEROSOL PREPARATION UNDER SAID PRESSURE AT ROOM TEMPERATURE FOR A PERIOD OF 1 TO 4 WEEKS. 